In the quantitative analysis of cholesterol and cholesterol esters which are present in the form of lipoproteins in body fluids such as blood, by using a dry type multilayer analysis element, blood, as an aqueous sample, is used without being diluted with water, physiological saline, a pH buffer, etc. Hence, the color reaction in the analysis element does not proceed rapidly. Further, substances in the body fluid (e.g., neutral fats in the blood), make accurate analysis difficult.
In the attempt to solve the above problem, Japanese Patent Application (OPI) Nos. 96378/78 and 85200/86 (corresponding English literatures are shown hereinafter) and CLINICAL CHEMISTRY, 28, 1159-1162 (1982) proposed the joint use of an alkyl phenoxy polyethoxy ethanol. (The term "OPI" indicates an unexamined published patent open to public inspection.) However, techniques proposed in these references have been found to have the following problems:
(1) Cholesterol esterase activity is inhibited. PA1 (2) During storage before use, the dry type multilayer analysis element has a strong tendency to absorb water. Hence, the enzyme activity in the element is decreased or lost, and the performance of the element is degraded.
CLINICAL CHEMISTRY, 20, 470-475 (1974) and Japanese Patent Application (OPI) No. 125796/75 describe that cholesterol esters are efficiently hydrolyzed in aqueous solution by using cholesterol esterase and bile acid or its salt in combination. When this technique is applied to the dry type multilayer analysis element, spreading of an aqueous sample, particularly blood (whole blood, plasma or serum) is poor on a porous spreading layer, and the accuracy of analysis decreases drastically.